Processes of apoptosis and cell proliferation in uterine myomas originating from reproductive and perimenopausal women.

We studied uterine myomas originating from females of reproductive age and from females of perimenopausal age. Uterine myomas represent benign tumors of the myometrium, and they develop frequently in women of reproductive age. The frequency of uterine myomas increases with age until women reach the menopause. The study included patients with a myomatous uterus, in the reproductive age or peri-menopausal age, independently evaluating small and large myomas. Myometrial alterations in their direct vicinity were evaluated independently of the myomas. The study included evaluation of immunolocalization of two index proteins which participate in myoma cells growth control: Ki-67 nuclear antigen and caspase 3. In women of reproductive age, both in small and large myomas, elevated immunostaining of Ki-67 was noted in parallel to low levels of caspase 3 staining, which indicated the ongoing process of proliferation. In women of peri-menopausal age with small or large myomas, no Ki-67 immunostaining was detected, while staining of caspase 3 manifested low levels. Proliferation in reproductive age women myomas is higher than in the peri-menopausal age.

The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.

Outside stenting of the vein graft decreases VEGF-A expression and induces significant down-regulation of VEGFR-1 in the intimal and medial layers after the re-endothelialization period.

BACKGROUND: Neointimal hyperplasia (NIH) in vein grafts implanted into the arterial system develops after re-endothelialization and is considered a significant risk factor of occlusion. Evidence suggests that VEGF-A expression with VEGFR-2 activation and/or VEGFR-1 down-regulation might be involved in inhibiting NIH formation. The aim was to assess whether a stented vein graft (SV) has an impact on VEGF-A and VEGFR-1 expression compared with non-stented vein grafts. MATERIAL/METHODS: Twelve sheep received a radial vein with an outside stent (SV) and a radial vein (RV) transplanted into their carotid arteries. The covering of the luminal surface of the SV and RV grafts by endothelium was 98.3% and 96.3%, respectively, at 6 weeks. From the 6th to 12th weeks after transplantation, the time course of total VEGF-A expression and VEGFR-1 expression were evaluated separately for the intima and media. RESULTS: VEGF-A and VEGFR-1 expression were significantly lower in the SV than in the RV group in the intima. In the media the SV grafts were associated with higher VEGF-A and VEGFR-1 expression at 6 and 8 weeks, but lower values were observed at weeks 10 and 12 compared with the RV grafts. Comparing the time courses of VEGF-A and VEGFR-1 expression in the intima and media with intimal/medial thickening in the SV and RV groups, negative correlations for the SV grafts were found. CONCLUSIONS: These findings indicate that outside stenting of the vein graft decreases VEGF-A expression and induces significant down-regulation of VEGFR-1 in the intima and media after the re-endothelialization.

A new external stent - intimal proliferation and apoptosis in the vein graft in the animal model.

BACKGROUND: We have previously showed that the extravascular dacron mesh stent wrapped around a vein graft and implanted into the arterial system prevented the hypertrophy of the graft's wall, impeded the overgrowth of the intima and decreased the proliferation rate of venous graft cellular elements. AIM: To determine the role of cellular proliferation and apoptosis in the process of remodelling in the stent in an animal model in a 12-week period. METHODS: Male sheep (n = 21) received by transplantation the hybrid graft (group 1) or carotid artery radial vein grafts (group 2). A hybrid graft was composed of a radial vein, collagen fibrin glue and highly flexible torlen/dacron mesh tubing. Grafts were retrieved on day 5, 9 and then week 4, 6, 8, 10, 12, respectively. A proliferation process was assessed using a Ki-67 antigen kit. The presence of apoptosis was detected using a TUNEL kit, strictly according to the manufacturer's manual. RESULTS: The number of proliferating cells has presented a decreasing trend in both groups, whereas the mean quantity of apoptotic cells increased over a 12-week period (p < 0.001) in both groups. Proliferation was more prominent during the first 5 weeks in both groups. The trend had a tendency to reverse during the last 7 weeks of observation. The ratio of proliferating to apoptotic cells differed between groups (1.6 vs. 1.9 on day 5 and 0.2 vs. 0.6 in week 12, in group 1 and group 2, respectively). No linear correlation between proliferation and apoptosis was observed (p > 0.05). CONCLUSIONS: Different kinetics in the trico hybrid graft group in comparison with the radial vein graft group was observed, with a more prominent cellular turnover in the trico hybrid graft. Apoptosis in an unprotected vein wall was overcomed by the proliferation process. In trico hybrid vein grafts, beneficial remodelling of the intimal layer was predominantly dependent on inhibition of intimal proliferation rather than the effect of changes of the apoptosis ratio. There was no linear correlation between proliferation and apoptosis in the investigated grafts.

The aromatase expression in myomas and myometriums of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female pathologies. Uterine smooth muscle myomas or fibromas are benign tumours which respond to hormones and their etiology induces wide interest. The myomas were found to contain aromatase and, in addition, cells of the myomas were found to synthesize estrogen. This study was conducted on patients with the myomas, in either generative age or in the perimenopausal period. Expression of aromatase was detected in patients of various age, with large or small uterine myomas, using an immunohistochemical technique. In addition expression of the enzyme was examined at the periphery of every myoma.

Immunohistochemical localization of telomerase in myomas and in uterine myometrium.

Telomerase activity could be a potential marker for the neoplastic process, because it is absent in normal cells and present in tumor cells. Immunohistochemical studies were conducted using samples obtained from 32 uterine myomas, each sample having a size of 3-4 cm and obtained from women between 35 and 45 years of age. These studies also concentrated on fragments of macroscopically unaltered myometrium, collected 3-4 cm from a uterine tumor. Immunohistochemistry was performed using antibody to the catalytic unit of telomerase (hTERT; clone 44F12, NCL-L-hTERT, Novocastra Laboratories, UK). This study aimed at detecting a possible presence of potentially neoplastic cells in the margins of healthy tissue, which was removed together with the primary tumor. The results were classified according to the number of telomerase-positive cells. Tumors of the first group had up to 50% telomerase-positive cells, while their content in the second group exceeded 50%. Our study demonstrated an almost two-fold increase in the number of telomerase-positive tumor cells compared with myometrial cells 3-4 cm from the tumor. Hopefully, investigating the presence of telomerase in both uterine myometrium and myoma could facilitate the diagnosis of the neoplastic process.

Effect of L-carnitine supplementation on xenobiotic-metabolizing hepatic enzymes exposed to methanol.

The study aimed to evaluate the effect of L-carnitine on hepatic cytochrome P450-dependent monooxygenases exposed to methanol. Male Spraque-Dawley rats were given methanol (1/4 LD50 and 1/2 LD50) together with L-carnitine (1g/kg body weight). The parameters of microsome electron transport chains I and II and the levels of CYP2E1, CYP2B1/2 and CYP1A2 were measured 8, 12, 24, 48, 72 and 96 h after exposure. L-carnitine did not affect cytochrome P450 but it significantly increased at 72 and 96 h NADPH-cytochrome P450 reductase. It stimulated cytochrome b5 at 48 and 96 h and NADH-cytochrome b5 reductase activity at 12, 72 and 96 h. Methanol, especially the lower dose, inhibited cytochrome P450 after 48 h, but the higher methanol dose inhibited NADH-cytochrome b5 reductase activity in this time. L-carnitine, combined with the lower dose of methanol, stimulated NADPH-cytochrome P450 reductase after 48 h and cytochrome b5 and NADH-cytochrome b5 reductase over the whole period of observation. L-carnitine stimulated CYP2B1/2 but not CYP2E1 and CYP1A2. Methanol stimulated CYP2E1 at 24 h, but CYP1A2 at 96 h in the studied doses. CYP2B1/2 was induced by the lower dose of methanol at 24 h but by the higher one at 96 h. When given together, L-carnitine and methanol (1/2 LD50) significantly stimulated CYP2E1 up to 170% at 24 h and 145% at 96 h.

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease.

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats.

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b(5) as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b(5) reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b(5) (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b(5) and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b(5) and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40+/-0.15 microg/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.

Immunolocalization and expression of kallistatin and tissue kallikrein in human inflammatory bowel disease.

The distribution of tissue kallikrein (TK) and its plasma inhibitor, kallistatin in plasma and intestinal tissue, was studied in patients with active ulcerative colitis (UC) and Crohn's disease (CD). TK was localized to goblet cells and kallistatin to epithelial cells of normal human intestine. Both proteins are visualized in macrophages inside granulomas in CD as well as in plasmocytes in both CD and UC. Intestinal tissue kallikrein (ITK) and kallistatin are significantly decreased in inflamed intestine compared to noninflammatory controls. TK mRNA is significantly decreased in intestinal biopsy samples from active UC patients compared with inactive patients or controls. Immunoreactive TK is present in plasma in very low concentrations in patients and did not differ in normal subjects. Plasma kallistatin was significantly decreased in patients with active disease compared to normal controls. Our data suggest that release of TK during inflammation plays a role in inflammatory bowel disease.

A new outside stent--does it prevent vein graft intimal proliferation?

OBJECTIVE: The saphenous vein subjected to arterial pressure stretches to its elastic limits and constitutes intimal hyperplasia. Sheathing of the vein graft with pressure-resistant tubing might prolong vein graft patency. METHODS: Twenty-one sheep received radial vein grafts or hybrid grafts composed of radial vein, collagen fibrin glue and highly flexible torlen/dacron mesh tubing transplanted into the carotid artery position. Veins were examined with the use of light and electron microscopy. Proliferating cell antigen (Ki-67) stains served as markers of proliferation. RESULTS: The mean wall thickness of both intimal and medial layers was evaluated. The mean intimal wall thickness was 19+/-11 microm in hybrid grafts vs. 24+/-7 microm in unsheathed grafts (P<0.001); 22+/-6 vs. 26+/-10 microm (P<0.001); 23+/-8 vs. 52+/-15 microm (P<0.001); 37+/-21 vs. 90+/-31 microm (P<0.001); 57+/-31 vs. 104+/-28 microm (P<0.001); 58+/-21 vs. 133+/-32 microm (P<0.001); and 72+/-22 vs. 244+/-100 microm (P<0.001) after respectively 5 days, 9 days, 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks from implantation. Electronic microscope examination of hybrid grafts revealed a smooth endothelial layer with intact nuclei and an intima composed of layers of collagen and muscle fibers. In unsheathed grafts endothelial edema and nuclear destruction were observed. CONCLUSIONS: The external vein graft support with mesh tubing reduces intimal and medial layer thickening and cell proliferation in composite vein grafts transplanted in the arterial position.